質量分離と人間住血吸虫マンソン住血吸虫の Intramolluscan の段階の体外培養

In this video, we describe a detailed protocol for the large scale hatching and isolation of free swimming Schistosoma mansoni miracidia and the subsequent introduction into in vitro culture. Schistosoma mansoni is one of four major blood fluke species that infect over two hundred million people worldwide causing the neglected tropical disease known as Schistosomiasis. The schistosome life cycle is complex involving snails as intermediate hosts and mammals including humans as the definitive hosts.

In vitro cultivation of larval and adult schistosomes represents an invaluable tool for advancing research towards control and elimination of this parasitic disease. In this video, using a mouse model to produce S.mansoni eggs containing the snail-infected miracidial stage, we illustrate a step by step procedure for mass isolation of miracidia hatched from eggs recovered from livers and their in vitro cultivation. Assisting me in this demonstration is Nathalie Dinguirard and Codie Heinemann.

The following is a workflow diagram illustrating the three major steps to be covered in the accompanying video. In the first segment, we illustrate the processing of infected livers. Using mice infected with Schistosoma mansoni adults, we will describe how to remove their infected livers and the steps used to prepare them for tissue blending including removing a non-liver tissue and subsequent washing in sterile saline solution.

In the second segment, we will show how eggs are extracted from livers and miracidia are isolated. Eggs are released from livers by mechanical blending followed by the repeated washing of the homogenized liver tissue in saline by centrifugation. Pelleted liver tissue containing eggs are then re-suspended in sterile artificial pond water and carefully transferred to foil covered volumetric flasks.

Excess foam and floating liver debris is eliminated by the rapid sequential removal and addition of approximately 12 mills of pond water to the flask. After the last wash, pre-warmed pond water is gently added to the flask followed by illumination of the top few centimeters of the flask neck with the bright light source to attract miracidia. Once the miracidia have accumulated at the light source, in the third and final segment of the video, we will demonstrate the collection of these larvae and their cultivation.

Miracidia are transferred from the flask to 15 milliliters centrifuged tubes. Then immobilized on ice and concentrated by centrifugation. In the final steps, miracidia are pulled into a single tube.

The pond water is replaced with Chernin's balanced salt solution and miracidia are allocated into wells of the 24 well tissue culture plate for in vitro cultivation at 26 degrees centigrade. After euthanizing infected mice following institutional animal care approved protocols, they are placed on their back and 70%ethanol is sprayed on the ventral surfaces. To remove the liver, pinch and pull up skin of the lower abdomen and cut across the pinched skin with sterile surgical scissors.

Pull the skin up to reveal the liver and gently remove it by hand. Livers from infected mice appear enlarged and speckled due to an egg-induced granulomatous response. Once the liver's removed, place it in a beaker containing 200 mills of sterile 1.2%saline solution containing Pen Strep and proceed to remover livers from the remaining mice.

Livers are then transferred to a sterile Petri dish and any non-liver fat and connective tissues are removed with sterile forceps. Once the trimming is complete, livers are transferred to sterile 250 milliliter centrifuge bottles containing approximately 200 mills of sterile saline and vigorously shaken to suspend the extraneous tissues and excess blood. Using a sterile disposable pipette, remove the surface foam and any floating tissue debris.

Recap the bottle and vigorously shake a second time followed by, again, removing foam and debris and then carefully decanting and discarding the saline wash solution. Add 200 milliliters of fresh saline solution to the livers and repeat the process three more times. You should see a noticeable clearing of the discarded wash solution.

Place the washed livers into a small sterile stainless steel blender cup and add approximately two mills of saline with antibiotics. Cover the cup with foil and a Petri dish to prevent spills and blend for one minute by alternating low and high speeds. After blending, evenly distribute the liver suspension into sterile 250 milliliter centrifuged bottles.

Typically, four bottles are used when processing 20 livers. Fill each bottle with approximately 200 milliliters of sterile saline. Weigh or balance the bottles and shake each one prior to centrifuging the blended livers.

Following centrifugation, gently decant and discard the wash saline. You should observe the pelleted liver sediment containing the eggs at the bottom of the bottle and then repeat this step once more by adding 200 milliliters of fresh saline to each bottle and shaking vigorously to thoroughly re-suspend the liver sediment prior to centrifuging. During the centrifugation step, prepare two sterile one liter volumetric flasks by covering them completely with aluminum foil except for the top three centimeters.

Use separate pieces of foil to cover the body and neck of the flask to facilitate washing steps that will be described in the next segment. Okay, back to the livers. After centrifugation and discarding the last saline wash, add 200 milliliters of sterile artificial pond water containing Pen Strep to the pelleted liver tissue and again shake to completely re-suspend the sediment.

Carefully transfer the pond water liver suspension to the volumetric flasks that are now covered with aluminum foil. Using sterile pond water, fill up the flask including the neck and without delay, remove residual foam and floating liver tissue by quickly pipetting up approximately 12 mills of pond water containing debris and discarding it into a separate tube. Replace the fresh pond water and repeat this cleaning step three more times checking for the presence of miracidia in the discard tube.

Seize additional cleaning steps if miracidia appear swimming in the discard tube or when no more tissue debris is observed. After the last wash, slowly add eight milliliters of pre-warmed sterile pond water to create a temperature gradient at the top of the flask. This is best accomplished by slowly discharging water along the side of the flask neck to avoid mixing.

Place a small Petri dish to cover the flask opening and shine a bright light across the top of the neck above the foil cover to illuminate the upper surface of the water. Typically, within five to 10 minutes, swimming miracidia attracted to the light will concentrate in large numbers within the top two to three centimeters of the pond water surface. When miracidia have amassed at the surface, using a sterile pipette, remove and transfer approximately eight mills of pond water to a sterile 15 mill centrifuged tube.

Place the tube containing miracidia on ice. Add eight mills of warm, sterile pond water along the side of the volumetric flask and wait for another 10 minutes. Repeat the collection of miracidia three more times using new 15 mill tubes each time.

After the fourth collection, keep the final tube on ice for 10 minutes to allow immobilized miracidia to settle. This first set of isolated parasites typically represents approximately 80%of the total miracidial harvest. Pellet miracidia by centrifuging tubes for one minute in a pre-cooled refrigerated centrifuge.

Following centrifugation, immediately remove most of the supernatant, being careful not to disturb the parasite pellets. This step should be done quickly as miracidia will soon begin to start swimming. Gently re-suspend the miracidia and transfer them to a single 15 mill tube.

Rinse each of the original tubes with approximately one mill of sterile pond water and add to the pooled tube. Allow parasites to settle on ice for about five minutes and again, pellet them by centrifugation. Carefully remove the supernatant without disturbing the pellet and add six to eight milliliters of sterile Chernin's balanced salt solution or CBSS containing antibiotics.

Gently re-suspend the miracidia in CBSS and allocate them into a 24 well tissue culture plate. Incubate parasites at 26 degrees centigrade under normoxic conditions. As you can see, miracidia will actively swim in wells following initial incubation and most will start to settle at the bottom of the well to begin transforming to the primary sporocyst stage.

Larvae will start shedding the ciliated epidermal plates after two to three hours in CBSS culture. After 24 hours, the vast majority of miracidia will have shed their plates and completed their transformation to sporocysts. In vitro cultivation of parasitic elements, especially those of medical and veterinary importance, represents an invaluable research tool, not only for identifying and characterizing unique pathways, regulating growth, development, reproduction, and host immune interactions, but also by providing practical methods for screening identified molecules and pathways for potential therapeutic intervention.

Looking to the future with development and continual refinement of tools currently available for manipulating genes and gene expression in vitro through transgenic RNA interference and genome editing approaches, it is envisioned that efficient methods for large scale isolation and cultivation of miracidia from other important schistosome species, for example, liver flukes or lung flukes will be needed to further research advancements in this field. We anticipate that the basic approach for the isolation of larval schistosomes presented here could be easily adapted to accommodate these and other helmin species.